Bring to room temperature and strip
foil from required number of wells.
Empty wells and tap dry onto tissue.
Add 10µl of standard or sample
to each well.
Add 200µl progesterone-enzyme
label.
(Blank well is 0 standard and wash
buffer only)
Leave 2-2.5 hours at room
temperature (22oC).
Wash 3 times in wash buffer
(200µl; 2 min each). Tap dry on
paper each time.
*Add 200ul alkaline phosphatase
substrate
Leave 20 to 30 minutes.
Compare colour development of
samples with standards.
Read optical density at 570 nm after
zeroing machine on blank well and
compute concentration from standard
curve. Results are in ng
progesterone/ml plasma. (Alternative
substrate Sigma 104 at 140 mg/25ml
using 405 nm filter).
Standards
Progesterone in plasma from an
anoestrous animal - store frozen. Mix thoroughly after thawing.
WARNING
All solutions contain thimerosal
(0.01% w/v). This is toxic. Dispose
of carefully and wash down sinks
with excess water. Keep away from
food and children and wash your
hands after use.
*Dissolve
substrate tablet in 25ml substrate
buffer 2. Sufficient for one plate.
Store in the dark. Do not
contaminate with progesterone-enzyme
label 1. Takes 20-30 minutes to
dissolve.
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